Intranasal Netrin-1 modRNA-Loaded Extracellular Vesicles Reduce Neuronal Apoptosis after Ischemic Stroke

Ischemic stroke remains a leading cause of mortality and long-term neurological disability worldwide, while effective neuroprotective therapies remain limited. Netrin-1 is a multifunctional guidance molecule with neuroprotective properties, and chemically modified mRNA (modRNA) offers a promising platform for transient therapeutic protein expression. However, safe and efficient delivery of modRNA to the ischemic brain remains a major barrier to clinical translation. Here, we engineered Netrin-1 modRNA-loaded extracellular vesicles (EV@mNetrin-1) derived from human umbilical cord mesenchymal stem cells (hUMSCs) and administered them via a noninvasive intranasal route. EV-mediated delivery enabled intracellular translation of Netrin-1 modRNA into protein in neurons and increased Netrin-1 expression in ischemic brain tissue. In vitro, EV@mNetrin-1 significantly reduced neuronal apoptosis and improved neuronal survival following oxygen-glucose deprivation/reoxygenation (OGD/R). In vivo, intranasal administration of EV@mNetrin-1 enhanced brain Netrin-1 expression, reduced infarct volume, improved neurological outcomes, and attenuated neuronal apoptosis in transient middle cerebral artery occlusion (tMCAO) mice. Mechanistically, the neuroprotective effects of EV@mNetrin-1 were associated with DCC-dependent activation of PI3K/AKT signaling. Collectively, these findings demonstrate the therapeutic potential of intranasal EV-mediated Netrin-1 modRNA delivery for ischemic stroke and support the development of RNA therapeutics for central nervous system disorders.